Project Abstract Small molecule and protein signals provide a rich vocabulary for cellular communication. The production and consequences of these signals are exquisitely sensitive to cellular context and microenvironment. For example, synthesis of pro-inflammatory, anti-inflammatory, and pro-resolution oxylipins in response to environmental exposures or wounding is tightly controlled by immune cells that can shift oxylipin production on the minute timescale as the immune response progresses in real time. Furthermore, the same signaling molecule can bring about an entirely different downstream biological response depending on microenvironmental context. Dissecting the molecular dialogue between cell types is challenging, and new methods are required to address fundamental questions: What is the downstream biological function of each signaling molecule? How is the biological function different when molecules are present in mixtures? How do microbes ? like the bacteria and fungi present in our bodies ? affect the molecular landscape? Our lab is developing new tools to probe these questions including (1) microscale co- and multiculture methods that enable precise positioning of cell types to study signaling, (2) integration of microculture with small molecule extraction methods for downstream metabolomics analysis using mass spectrometry, (3) specialized culture platforms and extraction methods to isolate signals from complex human-bacteria-fungal multikingdom culture, and (4) novel cell-based behavioral assays to probe the effects of chemical signals on biological function (including angiogenesis, mucus production, and immune cell function). The present proposal expands our lab?s capabilities in areas (1) and (4). This proposal will create innovative functional assays to study vasodilation (blood vessel expansion, a hallmark of inflammation) and fibroblast myodifferentiation (which leads to harmful fibrosis and remodeling in chronic inflammation). Central to this proposal is the use of ?open? microfluidics and spontaneous capillary flow to sculpt gel structures in three dimensions with sub-millimeter precision. Our lab has made significant advances in directing gel flows using open microfluidics, resulting in user-friendly, cost-effective methods to perform microscale multiculture experiments within standard well plates. The proposed work builds on our capabilities and embraces a significant engineering challenge: producing blood vessel mimics that can dynamically dilate and contract while being easy to multiplex in order to study large sets of signaling molecules. The proposed methods will enhance the understanding of the signals involved in detrimental prolonged inflammation, critical to the development of better therapies for numerous inflammatory conditions. Further, the bioengineering and microfluidic approaches developed will translate to other three dimensional biomimetic culture platforms.